Solute-phase electrochemical aptamer sensors for improved longevity and sensitivity

ABSTRACT

A device and method for detecting the presence of, or measuring the concentration or amount of, at least one analyte in a sample fluid. The device 100 includes at least one electrode 150, a sensor fluid 18, a plurality of aptamers freely diffusing in the sensor fluid, and a plurality of redox tags associated with at least a subset of aptamers of the plurality of aptamers. The sensor fluid is capable of having a sample fluid 14 introduced thereinto, and the detection or measurement of any analyte may occur through a change in electron transfer from at least one redox tag of the plurality of redox tags.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. PatentApplication Ser. No. 63/082,834, filed on Sep. 24, 2020; claims thebenefit of the filing date of U.S. Patent Application Ser. No.63/082,999, filed on Sep. 24, 2020; claims the benefit of the filingdate of U.S. Patent Application Ser. No. 63/083,029, filed on Sep. 24,2020; claims the benefit of the filing date of U.S. Patent ApplicationSer. No. 63/085,484, filed on Sep. 30, 2020; claims the benefit of thefiling date of U.S. Patent Application Ser. No. 63/122,071, filed onDec. 7, 2020; claims the benefit of the filing date of U.S. PatentApplication Ser. No. 63/122,076, filed on Dec. 7, 2020; claims thebenefit of the filing date of U.S. Patent Application Ser. No.63/136,262, filed on Jan. 12, 2021; claims the benefit of the filingdate of U.S. Patent Application Ser. No. 63/150,667, filed on Feb. 18,2021; claims the benefit of the filing date of U.S. Patent ApplicationSer. No. 63/150,677, filed on Feb. 18, 2021; claims the benefit of thefiling date of U.S. Patent Application Ser. No. 63/150,712, filed onFeb. 18, 2021; claims the benefit of the filing date of U.S. PatentApplication Ser. No. 63/150,856, filed on Feb. 18, 2021; claims thebenefit of the filing date of U.S. Patent Application Ser. No.63/150,865, filed on Feb. 18, 2021; claims the benefit of the filingdate of U.S. Patent Application Ser. No. 63/150,894, filed on Feb. 18,2021; claims the benefit of the filing date of U.S. Patent ApplicationSer. No. 63/150,944, filed on Feb. 18, 2021; claims the benefit of thefiling date of U.S. Patent Application Ser. No. 63/150,953, filed onFeb. 18, 2021; claims the benefit of the filing date of U.S. PatentApplication Ser. No. 63/150,986, filed on Feb. 18, 2021; and claims thebenefit of the filing date of U.S. Patent Application Ser. No.63/197,674, filed on Jun. 7, 2021, the disclosures of each of which areincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

This invention relates generally to aptamer sensors.

BACKGROUND OF THE INVENTION

This section is intended to introduce the reader to various aspects ofart that may be related to various aspects of the present invention,which are described and/or claimed below. This discussion is believed tobe helpful in providing the reader with background information tofacilitate a better understanding of various aspects of the presentinvention. Accordingly, it should be understood that these statementsare to be read in this light, and not as admissions of prior art.

Electrochemical aptamer sensors can identify the presence and/orconcentration of an analyte of interest via the use of an aptamersequence that specifically binds to the analyte of interest. Thesesensors include aptamers attached to an electrode, wherein each of theaptamers has a redox active molecule (redox couple) attached thereto.The redox couple can transfer electrical charge to or from theelectrode. When an analyte binds to the aptamer, the aptamer changesshape, bringing the redox couple closer to or further from, on average,the electrode. This results in a measurable change in electrical currentthat can be translated to a measure of concentration of the analyte.

A major unresolved challenge for aptamer sensors (particularly thosewhere the aptamers are bonded to the working electrode) is the lifetimeof the sensors, especially for applications where continuous operationis required (“continuous” referring to multiple measurements over timeby the same device). Such aptamer sensors are susceptible to degradationdue to, among other things, solutes in a fluid sample that arepotentially harmful to the sensor (such as nucleases that can degradethe aptamers, or fouling proteins such as albumin). Harmful solutes suchas these can reduce the operational life of the sensor, and thus anydevice including an aptamer sensor. Thus, to date, it has been difficultto provide electrochemical aptamer sensors with a lifetime that allowscontinuous sensing to take place over an extended period of time.Furthermore, for aptamer sensors where the aptamer is bonded to theelectrode, the flexibility of design is limited and often thesensitivity of the aptamer therefore suffers as a consequence. Devicesand methods that resolve these challenges for aptamer sensors areneeded.

SUMMARY OF THE INVENTION

Certain exemplary aspects of the invention are set forth below. Itshould be understood that these aspects are presented merely to providethe reader with a brief summary of certain forms the invention mighttake and that these aspects are not intended to limit the scope of theinvention. Indeed, the invention may encompass a variety of aspects thatmay not be explicitly set forth below.

Many of the drawbacks and limitations stated above can be resolved bycreating novel and advanced interplays of chemicals, materials, sensors,electronics, microfluidics, algorithms, computing, software, systems,and other features or designs, in a manner that affordably, effectively,conveniently, intelligently, or reliably brings sensing technology intoproximity with biofluid and analytes.

One aspect of the present invention is directed to device for detectingthe presence of, or measuring the concentration or amount of, at leastone analyte in a sample fluid. The device includes at least oneelectrode, a sensor fluid, a plurality of aptamers freely diffusing inthe sensor fluid, and a plurality of redox tags associated with at leasta subset of aptamers of the plurality of aptamers. The sensor fluid iscapable of having a sample fluid introduced thereinto, and the detectionor measurement of any analyte may occur through a change in electrontransfer from at least one redox tag of the plurality of redox tags.

Another aspect of the present invention is directed to a method fordetecting the presence of, or measuring the concentration or amount of,at least one analyte in a sample fluid. The method includes (1) bringinga sample fluid into contact with at least one aptamer of a plurality ofaptamers that are freely diffusing in a sensor fluid, wherein aplurality of redox tags are associated with at least a subset ofaptamers of the plurality of aptamers; and (2) detecting or measuring achange in electron transfer from at least one redox tag of the pluralityof redox tags.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and advantages of the disclosed invention will be furtherappreciated in light of the following detailed descriptions and drawingsin which:

FIG. 1A is a cross-sectional view of a device in accordance withprinciples of the disclosed invention.

FIG. 1B is a cross-sectional view of an alternate embodiment of a devicein accordance with principles of the disclosed invention.

FIG. 2A is a schematic showing a prior art portion of an aptamer sensordevice having a passivating layer and an aptamer attached to anelectrode.

FIG. 2B is a schematic showing the aptamer and passivating layerportions of the aptamer sensor device of FIG. 2A degrading over time.

FIG. 3 is a schematic of yet another embodiment of a device inaccordance with principles of the present invention.

FIG. 4 is a graph showing the effect of a membrane in an aptamer sensordevice on percentage of solute retention versus molecular weight of thesolute.

FIG. 5A is a schematic of yet another embodiment of a device inaccordance with principles of the present invention.

FIG. 5B is a schematic showing an alternate embodiment of an aptamerwith attached redox tag that can be used with devices in accordance withprinciples of the disclosed invention.

FIG. 5C is a schematic showing yet another alternate embodiment of anaptamer with attached redox tag that can be used with devices inaccordance with principles of the disclosed invention.

FIG. 5D is a schematic showing yet another alternate embodiment of anaptamer with attached redox tag that can be used with devices inaccordance with principles of the disclosed invention.

FIG. 5E is a schematic showing yet another alternate embodiment of anaptamer with attached redox tag that can be used with devices inaccordance with principles of the disclosed invention.

FIG. 6A is a schematic of yet another embodiment of a device inaccordance with principles of the present invention.

FIG. 6B is a schematic showing an alternate embodiment of an aptamerwith attached redox tag that can be used with devices in accordance withprinciples of the disclosed invention.

FIG. 6C is a schematic showing yet another alternate embodiment of anaptamer with attached redox tag that can be used with devices inaccordance with principles of the disclosed invention.

FIG. 7 is a schematic of yet another embodiment of a device inaccordance with principles of the present invention.

FIG. 8 is a schematic of yet another embodiment of a device inaccordance with principles of the present invention.

FIG. 9 is a schematic of yet another embodiment of a device inaccordance with principles of the present invention.

FIG. 10A is a graph showing raw chronoamperometric scans of currentversus time for cortisol in an exemplary device.

FIG. 10B is a graph showing normalized current gain for three sensorsversus concentration of cortisol.

DEFINITIONS

As used herein, “continuous sensing” with a “continuous sensor” means asensor that changes in response to changing concentration of at leastone solute in a solution such as an analyte. Similarly, as used herein,“continuous monitoring” means the capability of a device to providemultiple measurements of an analyte over time.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, pH, size, concentration orpercentage is meant to encompass variations of ±20% in some embodiments,±10% in some embodiments, ±5% in some embodiments, ±1% in someembodiments, ±0.5% in some embodiments, and ±0.1% in some embodimentsfrom the specified amount, as such variations are appropriate to performthe disclosed method.

As used herein, the term “electrode” means any material that iselectrically conductive such as gold, platinum, nickel, silicon,conductive liquid infused materials such as ionic liquids, PEDOT:PSS,conductive oxides, carbon, boron-doped diamond, nanotubes or nanowiremeshes, or other suitable electrically conducting materials.

As used herein, the term “blocking layer” or “passivating layer” means ahomogeneous or heterogeneous layer of molecules on an electrode whichalter the electrochemical behavior of the electrode. Examples include amonolayer of mercaptohexanol on a gold electrode or as another examplenatural small-molecule solutes in serum that form a layer on a carbonelectrode.

As used herein, the term “aptamer” means a molecule that undergoes aconformation or binding change as an analyte binds to the molecule, andwhich satisfies the general operating principles of the sensing methodas described herein. Such molecules are, e.g., natural or modified DNA,RNA, or XNA oligonucleotide sequences, spiegelmers, peptide aptamers,and affimers. Modifications may include substituting unnatural nucleicacid bases for natural bases within the aptamer sequence, replacingnatural sequences with unnatural sequences, or other suitablemodifications that improve sensor function, but which behave analogousto traditional aptamers. Two or more aptamers bound together can also bereferred to as an aptamer (i.e., not separated in solution). Aptamerscan have molecular weights of at least 1 kDa, 10 kDa, or 100 kDa.

As used herein, the term “redox tag” or “redox molecule” means anyspecies such as small or large molecules with a redox active portionthat when brought adjacent to an electrode can reversibly transfer atleast one electron with the electrode. Redox tag or molecule examplesinclude methylene blue, ferrocene, quinones, or other suitable speciesthat satisfy the definition of a redox tag or molecule. In some cases, aredox tag or molecule is referred to as a redox mediator. Redox tags ormolecules may also exchange electrons or change in behavior when broughtinto proximity with other redox tags or molecules.

As used herein, the term “change in electron transfer” means a redox tagwhose electron transfer with an electrode has changed in a measurablemanner. This change in electron transfer can, for example, originatefrom availability for electron transfer, distance from an electrode,diffusion rate to or from an electrode, a shift or increase or decreasein electrochemical activity of the redox tag, or any other embodiment astaught herein that results in a measurable change in electron transferbetween the redox tag and the electrode.

As used herein, the term “optical tag” or “fluorescent tag” means anyspecies that fluoresces in response to an optical source such as LED andwhose fluorescence is detectable by a photodetector such as aphotodiode. Example fluorescent tags include fluorescein and may be usedin combination with other fluorescent tags or optical quenchers such ablack-hole quencher dyes to change the fluorescence of the optical tag.

As used herein, the term “signaling aptamer” means an aptamer that istagged with a redox active molecule or tag and/or contains a redoxactive portion itself and which provides a change in electrochemicalsignal when it is released from an anchor aptamer.

As used herein, the term “anchor aptamer” means an aptamer that that canbind to a signaling aptamer, and when bound to the signaling aptamerchanges at least one property of the bound vs. unbound signaling aptamersuch as molecular weight, diffusion coefficient, charge state, beingfloating in solution vs. being immobilized, or some other property whichcauses a change in electron transfer with an electrode. The binding ofthe anchor aptamer with the signaling aptamer can be dependent onconcentration of the analyte to be measured.

As used herein, the term “folded aptamer” means an aptamer that alongits length associates with itself in one or more locations creating atwo or three-dimensional structure for the aptamer that is distinct froman “unfolded aptamer” that is a freely floating and oscillating strandof aptamer. Aptamers can also be partially folded or partially unfoldedin structure or in time spent in the folded vs. unfolded states.Multiple folding configurations are also possible.

As used herein, the term “analyte” means any solute in a solution orfluid which can be measured using a sensor. Analytes can be smallmolecules, proteins, peptides, electrolytes, acids, bases, antibodies,molecules with small molecules bound to them, DNA, RNA, drugs,chemicals, pollutants, or other solutes in a solution or fluid.

As used herein, the term “membrane” means a polymer film, plug ofhydrogel, liquid-infused film, tiny pore, or other suitable materialwhich is permiselective to transport of a solute through the membrane bysolute parameters such as size, charge state, hydrophobicity, physicalstructure, or other solute parameters than can enable permiselectivity.For example, a dialysis membrane is permselective by passing smallsolutes but not large solutes such as proteins. Membranes as understoodherein need not be multiporous, for example a nanotube or nanopore canact as a permiselective filter and is therefore considered part of amembrane as understood for the present invention. Permiselectivity canscale with the analyte, for example a membrane with a molecular weightcut-off of 50 kDa could be used to measure a 20-30 kDa protein but couldstill keep out cellular or other large content (globulins, fibrogen,etc.) and retain in aptamer that adequately large or physicallystructured such that permeability through the membrane is slow or nil.

As used herein, the term “sample fluid” means any solution or fluid thatcontains at least one analyte to be measured.

As used herein, the term “sensor fluid” means a solution or fluid thatdiffers from a sample solution by at least one property, and throughwhich the sensor solution and the sample solution are thereforeseparated but are in fluidic connection through at least one pathwaysuch as a membrane. The sensor solution comprises at least one aptameras a solute.

As used herein, the term “reservoir fluid” means a solution or fluidthat differs from a sample solution by at least one property, andthrough which the sensor solution and the reservoir solution are influidic connection through at least one pathway such as a membrane or apin-hole connection. A reservoir fluid may have multiple functions in adevice, for example, by introducing a solute continuously or as neededby diffusion equilibrium into the sensor fluid, or for example removingunwanted solutes from a sensor fluid and acting as a “waste removalelement”.

As used herein, a “device” comprises at least one sensor based on atleast one aptamer, at least one sensor solution, and at least one samplesolution. Devices can sense multiple samples and be in multipleconfigurations such as a device to measure a pin-prick of blood, or amicroneedle or in-dwelling sensor needle to measure interstitial fluid,or a device to measure saliva, tears, sweat, or urine sensor, or adevice to measure water pollutants or food processing solutes, or otherdevices which measure at least one analyte found in a sample solution.

DETAILED DESCRIPTION OF THE INVENTION

One or more specific embodiments of the present invention will bedescribed below. In an effort to provide a concise description of theseembodiments, all features of an actual implementation may not bedescribed in the specification. It should be appreciated that in thedevelopment of any such actual implementation, as in any engineering ordesign project, numerous implementation-specific decisions must be madeto achieve the developers' specific goals, such as compliance withsystem-related and business-related constraints, which may vary from oneimplementation to another. Moreover, it should be appreciated that sucha development effort might be complex and time consuming, but wouldnevertheless be a routine undertaking of design, fabrication, andmanufacture for those of ordinary skill having the benefit of thisdisclosure.

Certain embodiments of the disclosed invention show sensors as simpleindividual elements. It is understood that many sensors require two ormore electrodes, reference electrodes, or additional supportingtechnology or features which are not captured in the description herein.Sensors are preferably electrical in nature, but may also includeoptical such as a LED or laser excitation source and a photodetector,chemical, mechanical, or other known biosensing mechanisms. Sensors canbe in duplicate, triplicate, or more, to provide improved data andreadings. Sensors may provide continuous or discrete data and/orreadings. Certain embodiments of the disclosed invention showsub-components of what would be sensing devices with more sub-componentsneeded for use of the device in various applications, which are known(e.g., a reference or counter electrode, a battery, antenna, adhesive),and for purposes of brevity and focus on inventive aspects, suchcomponents may not be explicitly shown in the diagrams or described inthe embodiments of the disclosed invention. All ranges of parametersdisclosed herein include the endpoints of the ranges.

As described above, one aspect of the present invention is directed todevice for detecting the presence of, or measuring the concentration oramount of, at least one analyte in a sample fluid. The device includesat least one electrode, a sensor fluid, a plurality of aptamers freelydiffusing in the sensor fluid, and a plurality of redox tags associatedwith at least a subset of aptamers of the plurality of aptamers. Thesensor fluid is capable of having a sample fluid introduced thereinto,and the detection or measurement of any analyte may occur through achange in electron transfer from at least one redox tag of the pluralityof redox tags.

Another aspect of the present invention is directed to a method fordetecting the presence of, or measuring the concentration or amount of,at least one analyte in a sample fluid. The method includes (1) bringinga sample fluid into contact with at least one aptamer of a plurality ofaptamers that are freely diffusing in a sensor fluid, wherein aplurality of redox tags are associated with at least a subset ofaptamers of the plurality of aptamers; and (2) detecting or measuring achange in electron transfer from at least one redox tag of the pluralityof redox tags.

With reference to FIGS. 1A and 1B, exemplary embodiments of devices inaccordance with principles of the disclosed invention are shown.Referring first to FIG. 1A, a device 100 is shown as being placedpartially in-vivo into the skin 12 of a subject. Skin 12 includes theepidermis 12 a, the dermis 12 b, and the subcutaneous or hypodermis 12c. The device 100 includes a feature 112 that allows for access tosample fluids from the subject. Such sample fluids may includeinterstitial fluid (from the dermis 12 b) and/or blood (from a capillary12 d). In the embodiment shown in FIG. 1A, the feature 112 includes aplurality of microneedles (which may be formed of metal, polymer,semiconductor, glass, or other suitable material). Each of themicroneedles 112 projects from a first substrate 108. And eachmicroneedle 112 may include a hollow lumen 132. The device 100 alsoincludes a second substrate 110 (which may be a material such as polymeror glass) having an electrode 150 adjacent thereto. An optionalpassivating layer 120 may be adjacent to electrode 150, such thatelectrode 150 is positioned between passivating layer 120 and secondsubstrate 110. Passivating layer 120 includes a compound such asmercaptohexanol or may comprise natural solutes that have diffused intothe device 100 from the dermis 12 b.

As can be seen in FIG. 1A, a defined volume 130 is present between firstsubstrate 108 and passivating layer 120. It will be recognized by thoseof ordinary skill in the art that defined volume 130 does notnecessarily have to be defined by first substrate 108 and passivatinglayer 120—and in embodiments where passivating layer is absent, volume130 may be defined by first substrate 108 and electrode 150; or,alternatively, may be defined by first substrate 108 and secondsubstrate 110. A sensor fluid 18 may be present within volume 130 (asshown in FIG. 1A). Further, as can be seen in the embodiment of FIG. 1A,at least one membrane 136 is present between first substrate 108 andpassivating layer 120, and is positioned adjacent first substrate 108.The at least one membrane 136 may be of various materials orsubstances—such as a dialysis membrane or hydrogel, for example. In theparticular embodiment shown in FIG. 1A, portions of the membrane 136overlie the boundary between volume 130 and lumens 132 of eachmicroneedle 112. Due to this positioning of membrane 136, volume 130includes sensor fluid 18, and lumens 132 include sample fluid 14—such asinterstitial fluid from dermis 12 b or blood from capillary 12 d.Together the total volume provided by volume 130 and lumens 132 can be amicrofluidic component such as channels, a hydrogel, or other suitablematerial. A diffusion or other fluidic pathway exists from the samplefluid 14, such as interstitial fluid or blood, into volumes 132, 130.

Another embodiment of a device 100 is shown in FIG. 1B. This embodimentalso includes first and second substrates 108, 110, microneedles 112having lumens 132, electrode 150, passivating layer 120, defined volume130 and at least one membrane 136. As can be seen from FIG. 1B, in thisembodiment, electrode 150 and passivating layer 120 are recessed insecond substrate 110 (as opposed to the configuration shown in FIG. 1A).Thus, volume 130 is defined by first substrate 108 and combination ofsecond substrate 110 and passivating layer 120. Further, the embodimentas shown in FIG. 1B includes a plurality of membranes 136, with eachmembrane 136 positioned in a distal end of each lumen 132 of eachmicroneedle 112. Due to this positioning of membranes 136, both volume130 and lumens 132 include a sensor fluid 18 (and sample fluid ispresent in, and obtainable from, dermis 12 b and capillary 12 d, forexample).

Alternative arrangements and materials to those discussed above withrespect to FIGS. 1A and 1B are possible, such as using a single needleor hydrogel polymer microneedles. In addition, one or more of thefeatures of device 100 or the entire device 100 could be implanted intothe body and perform similarly as described herein. Furthermore, adevice 100 could be fully outside the body, if for example sampling afluid such as sweat or tears.

Turning now to FIGS. 2A and 2B, where like numerals refer to likefeatures, a portion of a prior art device 200 is shown. Referring toFIG. 2A, an aptamer sensor includes a passivating or blocking layer 248(including a compound such as mercaptohexanol) attached to an electrode250 (made from a material such as gold), and having at least one aptamer270 that is attached to the electrode 250, such as by being thiol-bondedto the electrode 250. The aptamer 270 has at least one redox tag ormolecule 240, such as methylene blue, associated therewith. The device200 is shown as being positioned in a sample fluid 14, such as blood orinterstitial fluid (for example). This prior art device 200 may have ananalyte (not shown) that binds with the aptamer 270, thereby changingthe availability of the redox tag 240 to the electrode 250, such as bybringing it closer to, or further from, the electrode 250. Conventionalaptamer sensors can be limited in performance because an aptamer that isbound to an electrode often has a weaker binding affinity to an analytethan an aptamer that is free in solution. In addition, as shown in FIG.2B, the sensors can degrade as the aptamer 270 and/or blocking layer 248degrades over time (e.g., chemical degradation, or detaching from theelectrode 250). Also, because such prior art devices 200 have relied onexogenous molecules (e.g., mercaptohexanol) for passivation, thepassivation layer 248 can also become thicker with fouling from solutes(such as albumin) in the sample fluid 14.

Thus, and with reference now to FIG. 3 , where like numerals refer tolike features, an embodiment of the disclosed invention that improves onthe prior art devices and reduces or eliminates drawbacks with suchdevices is shown. To that end, FIG. 3 shows a device 300 (or at least aportion thereof) that includes an electrode 350 and at least onemembrane 336 which separates a sample fluid 14 from a sensor fluid 18.The sensor fluid 18 contains a plurality of aptamers 370 having redoxtags 340. The electrode 350 may include a passivating layer 348. Thepassivating layer 348 may comprise one or more endogeneous solutes 16from the sample fluid 14 itself (or, as initially prepared, thepassivating layer 348 may be prepared from molecules that are known tobe endogenous to the sample fluid to be tested). Examples of suchendogenous molecules 16 include small molecules such as amino acids,hormones, metabolites, or peptides. (Thus, the device 300 shown in FIG.3 differs from that described above in prior art FIGS. 2A and 2B in thatthe prior art device described above included an aptamer and anexogenous molecule, such as mercaptohexanol. Similarly, electrode 350could also contain a passivation layer 348 comprised, at least in part,by an exogenous molecule such as hexanethiol or mercaptohexanol. But,even in that case, the passivation layer could detatch from theelectrode 350 and be in need of replacement.) By including endogeneousmolecules 16 in the passivation layer 348, longer lifetime of the device300 is achieved because endogenous molecules 16 can leave the electrode350 as shown by arrow 392 and another endogenous molecule 16 can replacethat now-missing molecule as shown by arrow 390. Thus, in a sense, thevery molecules in the sample fluid 14 can be used to “repair” thepassivation layer as it degrades, thereby extending the life of thedevice. (As mentioned above, these endogenous molecules can originatefrom the sample fluid itself, be already present as a deliberatecomponent of the sensor fluid, or could be a mix of the two.) As anon-limiting example, membrane 336 is able to pass in small solutes(e.g., <1 kDa)—for example, an analyte such as cortisol—and passivatingsolutes 16, such as amino-acids and peptides, but retains the aptamer370 (with redox tag 340) which is often >10 kDa in molecular weight. Ifthe aptamer 370 with redox tag 340 were not retained by the membrane336, then aptamer 370 with redox tag 340 could be lost into the body andno longer able be able to provide a measurement of the analyte.

An example of the analysis of the use of a membrane to pass smallsolutes (small target analyte) while retaining aptamers within device isshown with reference to FIG. 4 , which shows an illustrative plot ofsolute retention for a membrane such as membrane 336. This is an exampleonly, and shows that if measuring a small analyte such as cortisol (<400Da) and using a large aptamer (>10 kDa or even >50 kDa) a membrane couldbe highly permeable to the analyte and poorly permeable to the aptamer.Thus, for example, in various embodiments, membranes of the presentinvention may have molecular weight cutoffs (i.e., the molecular weightabove which a molecule will not easily pass through the membrane) thatare at least one of <300 Da, <1000 Da, <3 kDa, <10 kDa, <30 kDa, <100kDa, <300 kDa. Larger molecular weight cut-off membranes will requirelarger sized aptamers to prevent the aptamers from potentially escapingthe device.

Several additional embodiments will be discussed below. In theseadditional embodiments, an increase in availability of the redox tag tothe electrode can occur as a result of aptamer binding analyte, or,alternatively, without aptamer binding to an analyte. And even thougheach of the embodiments discussed below (and their respective figures)may show one specific example, the other the embodiments of theinvention are not so limited (e.g., the various aptamer/redox tag typescan be used across the various embodiments of devices disclosed herein,and vice versa.

Turning now to FIGS. 5A-5E, where like numerals refer to like features:FIG. 5A shows a portion of a device 500 including a substrate 510, asensor fluid 18, a plurality of aptamers 570 with redox tags 540 free inthe sensor fluid 18, a passivation layer 548 of endogenous solutes 16,and an electrode adjacent the substrate 510. Though not part of thedevice, analytes 19 are also depicted as present in the sensor fluid ofthe device. The schematic shown in FIG. 5A also depicts an electrontransfer event that occurs between a redox tag 540 and the electrode550. This is shown generally at reference numeral 598, and is anon-limiting example depicting that electron transfer 598 from a redoxtag 540 occurs in an increased amount, or frequency, or rate, whenaptamer 570 binding to analyte 19 occurs (e.g., as shown in the figure,when analyte is not bound to aptamer, the redox tag is not available—oris less available—to the electrode, due to, for example, a conformationof the aptamer that hinders or prevents such transfer when not bound toanalyte; conversely, when aptamer binds analyte, the conformation ofaptamer may change in a manner that positions the redox tag for electrontransfer). In various embodiments, aptamer binding to analyte canprovide changes in electron transfer and redox current (compared tobaseline transfer and current—i.e., transfer/current in the absence ofanalyte binding) of greater than 5%, greater than 10%, greater than 20%,greater than 50%, greater than 100%, or greater than 200%. For theembodiments illustrated herein, non-limiting examples of electricalmeasurement techniques may include voltammetry, square wave voltammetry,amperometry, chronoamperometry, coulometry, chronocoulometry, with apreferred embodiment being square wave voltammetry.

FIG. 5B schematically depicts another example of an aptamer 570 withattached redox tag 540 (that differs from the aptamer 570/redox tag 540schematically shown in FIG. 5A). The embodiment of the aptamer 570 inFIG. 5B is designed such that the redox tag 540 is more available forelectron transfer with the electrode 550 in the absence of any analyte19 binding to the aptamer 570 (high electron transfer—or high ET).Conversely, when analyte 19 binds to the aptamer 570 of FIG. 5B, theredox tag 540 is less available for electron transfer with the electrode550 (e.g., the redox tag 540 is less exposed—low ET).

FIG. 5C schematically depicts yet another example of an aptamer withattached redox tag 540 (that differs from the aptamer 570/redox tag 540schematically shown in FIGS. 5A and 5B). The embodiment shown in FIG. 5Cis designed with two aptamer portions: a signaling aptamer 572 and ananchor aptamer 574. A redox tag 540 is associated with (such as by beingattached to) the signaling aptamer 572. The anchor aptamer 574 includesa portion that has affinity for, and thus can bind, analyte 19. Whenanalyte 19 is not bound to the anchor aptamer 574 (left side of FIG.5C), the signaling aptamer 572 remains associated with the anchoraptamer 574, and so the redox tag 540 on signaling aptamer 572 is lessavailable for electron transfer with the electrode 550 (low ET).However, once the anchor aptamer 574 binds to analyte 19 (right side ofFIG. 5C), signaling aptamer 572 is released from anchor aptamer 574, andthe redox tag 540 becomes more available for electron transfer with theelectrode 550 (high ET). It will be recognized that the device of theembodiment of FIG. 5C has a plurality of aptamers—and thus includes aplurality of signaling aptamers 572, and a plurality of anchor aptamers574. As described above, each anchor aptamer of the plurality of anchoraptamers is adapted to bind to analyte. In one embodiment, eachsignaling aptamer of a majority of the plurality of signaling aptamersis bound to a respective anchor aptamer when a majority of anchoraptamers are not bound to any analyte. (This may occur, for example,prior to the introduction of any analyte.) Once analyte is introduced(such as when a sample fluid is introduced into the device—e.g., bybeing introduced into the sensor fluid of the device), at least a subsetof anchor aptamers from the plurality of anchor aptamers then binds toanalyte. When this occurs, a subset of signaling aptamers (from thetotal plurality of aptamers) dissociates from the anchor aptamers—andthe redox tag becomes more available for electron transfer with theelectrode.

Further, while the embodiment shown in FIG. 5C depicts analyte 19binding to anchor aptamer 574 and redox tag 540 on signaling aptamer572, in an alternate embodiment analyte binding may occur with signalingaptamer (signaling aptamer having redox tag), and binding of analyte tosignaling aptamer may serve to release signaling aptamer from anchoraptamer (such as by change in conformation of signaling aptamer). Evenfurther configurations as possible, as understood by those skilled inthe art of aptamers. To maximize the signal gain (change in signal)signaling aptamer 572 concentration will typically be less than or equalto the anchor aptamer 574 concentration else the signaling aptamer cancause increased background signal with or without the presence ofanalyte.

FIG. 5D schematically depicts yet another example of an aptamer withattached redox tag 540 (that differs from the aptamers/redox tagsschematically shown in FIGS. 5A, 5B, and 5C). The embodiment of theaptamer 570 in FIG. 5D has both a redox tag 544 and a redox quencher 542associated therewith (such as by being bound to the aptamer 570). Whenanalyte 19 is not bound to the aptamer 570, the redox tag 544 and redoxquencher 542 are spatially separated (left side of FIG. 5D) therebyallowing for greater electron transfer between redox tag 544 andelectrode 550 (high ET). However, once the aptamer 570 binds to analyte19 (right side of FIG. 5D), the redox tag 544 and redox quencher 542 arebrought into closer proximity with one another, thereby causing lesselectron transfer between redox tag 544 and electrode 550 (low ET).Numerous quenchers are possible, including anthraquinone-based redoxmolecules that can be self-quenching when two of such identicalmolecules are brought close together (monomer vs. dimer).

FIG. 5E schematically depicts yet another example of an aptamer withattached redox tag 540 (that differs from the aptamers/redox tagsschematically shown in FIGS. 5A, 5B, 5C and 5D). The embodiment of theaptamer 570 in FIG. 5E has both a first redox tag 546 and a second redoxtag 548 associated therewith (such as by being bound to the aptamer570). When analyte 19 is not bound to the aptamer 570, the first andsecond redox tags 546, 548 are spatially separated (left side of FIG.5E) thereby allowing for greater electron transfer between first andsecond redox tags 546, 548 and electrode 550 (high ET). However, oncethe aptamer 570 binds to analyte 19 (right side of FIG. 5E), the firstand second redox tags 546, 548 are brought closer together and theelectron transfer from one of the redox tags 546, 548 to the electrode550 is altered due to a two-step mediated electron transfer process, orother effect, for two redox tags brought into close proximity. Thesechanges in electron transfer are depicted in the voltammograms as shownas 546 a and 548 a. A non-limiting example of redox tags that enable theembodiment of FIG. 5E include methylene blue and ferricyanide.

Turning now to FIGS. 6A-6C, where like numerals refer to like features:FIG. 6A shows a portion of a device 600 that includes a substrate 610,at least first and second electrodes 650, 652, a passivation layer 648including endogenous solutes 16, a sensor fluid 18 (which, in theembodiment illustrated in FIG. 6A is inside an optional hydrogel 638), aplurality of aptamers 670 having redox tags 640 (free in solution), anda diffusion or iontophoretic pathway 694. Though not part of the device,analytes 19 are also depicted as present in the sensor fluid of thedevice. FIG. 6A also schematically depicts electron transfer that canoccur between redox tags 640 and the first and second electrodes 650,652. As can be seen in FIG. 6A, as a non-limiting example, electrontransfer 698 from the redox tags 640 in an increased amount, orfrequency, or rate, when analyte 19 is bound to the aptamer 670. Forexample, when analyte is bound to aptamer, the hydrodynamic radius orsize of the aptamer is smaller and therefore providing a fasterdiffusion coefficient, which results in the redox tag being moreavailable for electron transfer with the electrodes (such a version willbe discussed in greater detail below with respect to FIG. 6B); or, forexample, when analyte is not bound to aptamer, the redox tag is notavailable—or is less available—to the electrode, due to, for example, aconformation of the aptamer that hinders or prevents such transfer whennot bound to analyte; conversely, when aptamer binds analyte, theconformation of aptamer may change in a manner that positions the redoxtag for electron transfer. In various embodiments, aptamer binding toanalyte can provide changes in electron transfer and redox current(compared to baseline transfer and current—i.e., transfer/current in theabsence of analyte binding) of greater than 5%, greater than 10%,greater than 20%, greater than 50%, greater than 100%, or greater than200%. For the embodiments illustrated in FIGS. 6A-6C, non-limitingexamples of electrical measurement techniques may include voltammetry,square wave voltammetry, amperometry, chronoamperometry, coulometry,chronocoulometry, with a preferred embodiment being amperometry.

FIG. 6B schematically depicts another example of an aptamer 670 withattached redox tag 640 (that differs from the aptamer 670/redox tag 640schematically shown in FIG. 6A). The embodiment of the aptamer in FIG.6B is designed such that the redox tag 640 is less available forelectron transfer with the electrodes 650, 652 in the absence of analytebinding to aptamer (left side of FIG. 6B), because of a longer diffusiontime between the first and second electrodes 650, 652 where the analytecan undergo redox recycling (e.g. one electrode is a reducing electrode,one electrode is an oxidizing electrode). However, when analyte 19 bindsto the aptamer 670 (right side of FIG. 6B), the hydrodynamic radius orsize of the aptamer is smaller and therefore providing a fasterdiffusion coefficient, and therefore redox tag 640 is more available forelectron transfer with the first and second electrodes 650, 652. Thebinding of analyte 19 transforms the aptamer 670 between a long unfoldedaptamer 670 (in the absence of analyte 19 binding) and an aptamer 670with three stems when analyte 19 binds to aptamer 670.

As described above, with respect to FIG. 6A, a non-limiting example ofan environment within a device 600 may include an optional hydrogel. Insuch an embodiment, the hydrogel 638 (such as agar or polyacrylamide) isadded to further distinguish diffusion times between aptamers 670 boundto analyte 19 and aptamers 670 not bound to analyte. This is because thehydrogel 638 creates a more tortuous and size-selective diffusionpathway than a pure fluid would by itself. For example, an aptamer 670that fully dissociates could be modified to have a significant change inhydrodynamic radius (R), which changes its diffusion coefficient (D)according to D=kT/(6πηR). This equation is for diffusion in puresolution; a dense hydrogel 638 can be added to further distinguish thediffusion of the unfolded aptamer vs. the folded aptamer. The resultingcurrent between the redox recycling electrodes is proportional asI∞DC/z, where C is the concentration of the aptamer 670 and z theelectrode-to-electrode distance. With respect to changes in signal gain,the diffusion length of oglionucleotides (aptamers) varies with lengthto the ˜0.6^(th) power, and a 15 kDa protein that is globular/unfoldedcan have a change in R of 2.15/3.65.

FIG. 6C schematically depicts yet another example of an aptamer 670 withattached redox tag 640 (that differs from the aptamer 670/redox tag 640schematically shown in FIGS. 6A and 6B). The embodiment of the aptamerin FIG. 6C is designed with two aptamer portions: a signaling aptamer672 and an anchor aptamer 674. A redox tag 640 is associated with (suchas by being attached to) the signaling aptamer 672. The anchor aptamer674 includes a portion that has affinity for, and thus can bind, analyte19. When analyte 19 is not bound to the anchor aptamer 674 (left side ofFIG. 6C), the signaling aptamer 672 remains associated with the anchoraptamer 674, and so the redox tag 640 on signaling aptamer 672 is lessavailable for electron transfer with the first and second electrode 650,652 (as the combined signaling and anchor aptamers 672, 674 will exhibitslower diffusion in sensor solution and hydrogel). However, once theanchor aptamer 674 binds to analyte 19 (right side of FIG. 6C),signaling aptamer 672 is released from anchor aptamer 674, and the redoxtag 640 becomes more available for electron transfer with the first andsecond electrodes 650, 652 (as the liberated signaling aptamer 672 willexhibit more rapid diffusion in sensor solution and hydrogel). Further,while the embodiment shown in FIG. 6C depicts analyte 19 binding toanchor aptamer 674 and redox tag 640 on signaling aptamer 672, in analternate embodiment analyte binding may occur with signaling aptamer(signaling aptamer having redox tag), and binding of analyte tosignaling aptamer may serve to release signaling aptamer from anchoraptamer (such as by change in conformation of signaling aptamer).

It will be recognized that when the device shown in FIG. 6A uses theembodiment of aptamers of FIG. 6C, it will include a plurality ofsignaling aptamers 672, and a plurality of anchor aptamers 674. Asdescribed above, each anchor aptamer of the plurality of anchor aptamersis adapted to bind to analyte. In one embodiment, each signaling aptamerof a majority of the plurality of signaling aptamers is bound to arespective anchor aptamer when a majority of anchor aptamers are notbound to any analyte. (This may occur, for example, prior to theintroduction of any analyte.) Once analyte is introduced (such as when asample fluid is introduced into the device—e.g., by being introducedinto the sensor fluid of the device), at least a subset of anchoraptamers from the plurality of anchor aptamers then binds to analyte.When this occurs, a subset of signaling aptamers (from the totalplurality of aptamers) dissociates from the anchor aptamers—and theredox tag becomes more available for electron transfer with theelectrode.

With further reference to FIG. 6C, in addition to changes in diffusioncoefficient, the larger the effective sphere for the aptamer the lesslikely it will experience electron transfer with an electrode (with afirst principles estimation based on the inverse of sphere area,proportional to 1/R{circumflex over ( )}2). This example is simply toshow that two factors can be at play for embodiments of the presentinvention, both distance of the redox tag to the electrode and diffusiontime to/from the electrode. This diffusion time to an electrode appliesother embodiments as well, where for example with a chronoamperometricresponse for an aptamer the total current baseline could remain higheror reach baseline more quickly as diffusion coefficient for the aptamersincreases. This diffusion time to an electrode may also impactinterrogation methods such as square wave voltammetry, as aptamer thatis near the electrode can contribute to the signal as well if it is ableto diffuse to the electrode during each square window (during eachvoltage pulse that is applied). The first and second electrodes 650 and652 can be closely spaced via interdigitation or other suitabletechnique, and, in such an embodiment, may be within less than 50 μm,less than 10 μm, less than 2 μm, or less than 0.4 μm distant of eachother.

With reference to FIG. 7 where like numerals refer to like features,another embodiment in accordance with aspects of the present inventionis shown. As can be seen in FIG. 7 , a portion of a device 700 is shown,and includes a substrate 710, at least one electrode 750, a passivationlayer 748 including endogenous solutes 16, a sensor fluid 18, aplurality of aptamers having redox tags 740 (free in the sensorsolution), and a poorly-mobile or non-mobile material 738 in the sensorfluid 18. Though not part of the device, analytes 19 are also depictedas present in the sensor fluid of the device.

The aptamers/redox tags component of the embodiment of FIG. 7 is similarto that shown in FIGS. 5C and 6C, and includes two aptamer portions: asignaling aptamer 772 and an anchor aptamer 774. A redox tag 740 isassociated with (such as by being attached to) the signaling aptamer772. The anchor aptamer 774 includes a portion that has affinity for,and thus can bind, analyte 19. As can be seen in FIG. 7 , the anchoraptamer 774 is immobilized via linkage 739 to the poorly or non-mobilematerial 738. The poorly-mobile or non-mobile material 738 may comprisevarious materials, such as a hydrogel. In one non-limiting example, thematerial 738 could be a hydrogel such as polyacrylamide and the linkerbe a molecule such as acrydite that is attached to the anchor aptamer ata terminal end or other location. In an alternate embodiment, the anchoraptamer could be cross-linked with other anchor aptamers or the anchoraptamer made so large (e.g., >100 kDa) such that it is effectivelyimmobile in a dense hydrogel 738.

Still referring to FIG. 7 , when analyte 19 is not bound to the anchoraptamer 774, the signaling aptamer 772 remains associated with theanchor aptamer 774, and so the redox tag 740 on signaling aptamer 772 isless available for electron transfer with the electrode 750 (because thecombined signaling and anchor aptamers 772, 774 will be poorly-mobile ornon-mobile in the sensor fluid due to anchor aptamer 774 being linked tomaterial 738). However, once the anchor aptamer 774 binds to analyte 19,the signaling aptamer 772 is released from anchor aptamer 774 (asindicated by arrow 796), and the redox tag 740 becomes more availablefor electron transfer with the electrode 750 (because the liberatedsignaling aptamer 772 will exhibit more rapid diffusion in sensorsolution as it is no longer complexed with the anchor aptamer 774 thatis linked to poorly-mobile or non-mobile material 738). Further, whilethe embodiment shown in FIG. 7 depicts analyte 19 binding to anchoraptamer 774 and redox tag 740 on signaling aptamer 772, in an alternateembodiment analyte binding may occur with signaling aptamer (signalingaptamer having redox tag), and binding of analyte to signaling aptamermay serve to release signaling aptamer from anchor aptamer (such as bychange in conformation of signaling aptamer).

It will be recognized that the device of the embodiment of FIG. 7 has aplurality of aptamers—and thus includes a plurality of signalingaptamers 772, and a plurality of anchor aptamers 774. As describedabove, each anchor aptamer of the plurality of anchor aptamers isadapted to bind to analyte. In one embodiment, each signaling aptamer ofa majority of the plurality of signaling aptamers is bound to arespective anchor aptamer when a majority of anchor aptamers are notbound to any analyte. (This may occur, for example, prior to theintroduction of any analyte.) Once analyte is introduced (such as when asample fluid is introduced into the device—e.g., by being introducedinto the sensor fluid of the device), at least a subset of anchoraptamers from the plurality of anchor aptamers then binds to analyte.When this occurs, a subset of signaling aptamers (from the totalplurality of aptamers) dissociates from the anchor aptamers—and theredox tag becomes more available for electron transfer with theelectrode.

With reference to FIG. 8 , where like numerals refer to like features,another embodiment in accordance with aspects of the present inventionis shown. As can be seen in FIG. 8 , a portion of a device 800 is shown,and includes a substrate 810, at least one electrode 850, a membrane838, a sensor fluid 18, a plurality of aptamers having redox tags 740(free in the sensor fluid). Though not part of the device, analytes 19are also depicted as present in the sensor fluid of the device. Theaptamers/redox tags component of the embodiment of FIG. 8 is similar tothat shown in FIGS. 5C, 6C, and 7 , and includes two aptamer portions: asignaling aptamer 882 and an anchor aptamer 884. A redox tag 840 isassociated with (such as by being attached to) the signaling aptamer882. The anchor aptamer 884 includes a portion that has affinity for,and thus can bind, analyte 19. The membrane 838 exhibits selectivepermeability based on size, charge, or at least one solute property, andis able to pass a signaling aptamer 882 but not a signaling aptamer thatis attached to a larger anchor aptamer 884. Thus, the membrane 838impacts the availability of the redox couple 840 to the electrode 850.For example, a signaling aptamer could have a radius of 3 nm/2 nm infolded/unfolded states and an anchor aptamer have 27/7 nm infolded/unfolded state, creating a difference in size of ˜3-10× when asignaling aptamer is freed from an anchor aptamer. Nanofiltrationmembranes can provide is nM pore sizes, and ultrafiltration 10 s to 100s nm pore sizes (PES, track-etch, and other materials), resulting insize selective permeability that would enable mainly only the signalingaptamer 882 to penetrate the hydrogel or membrane 838.

And so, still referring to FIG. 8 , when analyte 19 is not bound to theanchor aptamer 884, the signaling aptamer 882 remains associated withthe anchor aptamer 884, and so the redox tag 840 on signaling aptamer882 is less available (or not available) for electron transfer with theelectrode 850 (because the signaling aptamer 882 will be unable to crossmembrane 838 due to being complexed with anchor aptamer 884). However,once the anchor aptamer 884 binds to analyte 19, the signaling aptamer882 is released from anchor aptamer 884 and is able to pass throughmembrane 838, resulting in the redox tag 840 becoming available forelectron transfer with the electrode 850. Further, while the embodimentshown in FIG. 8 depicts analyte 19 binding to anchor aptamer 884 andredox tag 840 on signaling aptamer 882, in an alternate embodimentanalyte binding may occur with signaling aptamer (signaling aptamerhaving redox tag), and binding of analyte to signaling aptamer may serveto release signaling aptamer from anchor aptamer (such as by change inconformation of signaling aptamer).

With reference to FIG. 9 , where like numerals refer to like features,another embodiment in accordance with principles of the presentinvention is shown. In certain of the various embodiments discussedherein, a membrane is used to selectively allow passage of certainmolecules and not of others. However, as no membrane is perfectly sizeselective, and as aptamers and redox tags can degrade over time, it maybe advantageous to continually introduce a fresh supply of aptamers,signaling aptamers, and/or anchor aptamers or other solutes thatincrease performance of the sensor or improve longevity of the sensor(e.g. nuclease inhibitors, for example). Thus, as shown in FIG. 9 , aportion of a device 900 includes substrates 910, at least one electrode950, a membrane 936, a sample fluid 14, a sensor fluid 18, and areservoir fluid 17. The membrane 936 exhibits mass flow represented atreference numeral 991, and the device also includes a diffusionrestrictive feature 935 (such as a pinhole or membrane) with a mass flowrepresented at reference numeral 993.

As a nonlimiting example of that shown in FIG. 9 , consider a 0.2 kDadialysis membrane for membrane 936 and assume the aptamers are 10-100×larger than the solute to be detected (e.g. phenylalanine, cortisol,etc.). Assume the system is designed such that the volume of reservoirfluid 17 is at least one of 2×, 10×, 50×, or 250× greater than volume ofsensor fluid 18 and that the mass flow 991 of aptamer is at least 2×,10×, 50×, or 250× less than mass flow of aptamer 993, while the massflow 991 of the analyte is at least 2×, 10×, 50×, or 250× greater thanthe mass flow of the analyte 993. As a result, the concentrations ofanalyte will be within at least 50%, 10%, 2%, or 0.4% of each other whencomparing sample fluid 14 with sensor fluid 18, and the concentrationsof aptamer will be within at least 50%, 10%, 2%, or 0.4% of each otherwhen comparing sensor fluid 18 and reservoir fluid 17.

As a geometrical example, consider a membrane 936 with 0.2 cm² area and10% porosity to the analyte, and a diffusion restrictive feature 935that is a pinhole in materials 910 and 950 0.001 cm² in area and 0.001cm in length. The mass transport for a small analyte through themembrane will be equivalent to 0.02 cm² area and the mass transportthrough the feature 935 0.001 cm², which is 20× different, satisfyingthe above stated criteria for design as shown in FIG. 9 . As a result,both analyte and aptamer concentrations can be maintained for prolongedperiods of times (days, weeks, months) even if aptamer is lost from thedevice or degraded over time. Aptamers could also degrade over time andtheir presence in the device and the presence of other contaminants suchas nucleases or proteins could be problematic. For example, if signalingaptamers became cleaved and their molecular weight decreased, they couldgive a false higher reading of signal in embodiments of the presentinvention. With membrane protection of the sensor fluid from the samplefluid, most degradation or contamination modes will be very slow, suchthat the reservoir may also act as a waste removal element.

The various embodiments disclosed herein can be enabled to beuser-calibrated, factory-calibrated, or calibration-free.User-calibration could for example require a pin-prick blood draw andrunning of a conventional assay to measure analyte concentration, andthat concentration data entered into the software that runs the sensingdevice.

Factory-calibrated implies that the device requires calibration, butthat the calibration is shelf-stable and stable for at least a portionof the use period of the device. Embodiments, such as those shown inFIG. 5B and FIG. 5C, could benefit from factory calibration if they areinterrogated by square wave voltammetry, and if the passivation layer548 thickness is kept fairly constant (e.g. using a mercaptohexanolpassivation layer 548 or polyethylene glycol terminated passivationlayer 548 that is resistant to fouling). In factory calibration, thedevice is tested with a sample fluid with a known concentration ofanalyte, and that information is then shipped along with the product inorder to enable it to start its use with proper calibration.

Calibration-free operation is possible if one could eliminate thefactors that could cause a sensor signal to drift or change. Consideringthe embodiments of FIGS. 6 and 9 , the aptamer concentrations can bekept constant, and with a chronoamperometric measurement response thechange in current vs. time will be dependent on diffusion coefficient ofthe signaling aptamer 672 vs. the anchor aptamer 674 and signalingaptamer 672 bound together. The diffusion coefficient will not change ina sample fluid such as interstitial fluid, and an overvoltage can besupplied to measure the chronoamperometric response even if thickness ofthe passivating layer 648 changes slightly. Simply, thechronamperometric response will measure the percentage of signalingaptamer 672 that is free from the anchor aptamer 674, which is directlyrelated to the binding affinity of the analyte to the aptamers, henceenabling calibration-free operation because the concentrations of thesignaling aptamers 672 and anchor aptamers 674 are known.Calibration-free operation is also possible using the constructs of FIG.5D or FIG. 5E by measuring changes in electron transfer rates, peakposition shifts, or ratios (not individual magnitudes) of two or moreredox peaks from different redox tags 546, 548 (FIG. 5E).

Although not described in detail herein, the embodiments of the presentinvention may also be applied to a simple single-use device. Forexample, device 100 of FIG. 1 could be applied to the skin 12, take asingle measurement of an analyte within 15 minutes of application of thedevice 100, and then remove the device 100 from the skin 12 surface.Alternately (not shown) a device format similar to a glucose test-stripor other device with a microfluidic channel or a wicking channel (like alateral flow assay) can be used to transport a sample fluid to one ormore sensors and embodiments as taught herein for the present invention.For example, the aptamers and redox tags could be dry and placed nearthe inlet of such a test strip and dissolved into the sample fluid andthen transported to a working electrode where measurement of the analyteis performed.

Although not illustrated in detail herein, the embodiments of thepresent invention may also be applied to a device with more than onetype of aptamer within a single device. For example, and referring toFIGS. 5A-5E, if an analyte had a very large dynamic range such ascortisol which can ranges from 1 s to 100 s of nM in free concentrationin blood or even higher in those with Cushing's syndrome, the range ofcortisol levels could be beyond the dynamic range of the device 500 ifonly a single type of aptamer was used for 570, 572, or 574. Instead,for example, half of the aptamers 570, 572, or 574 could have 100×weaker binding affinity for cortisol be used for a dynamic range of 0.5to 50 nM and the other half for 50 nM to 5000 nM. Together in a singledevice they could provide a dynamic range for sensing cortisol thatis >100× and as large as 10,000×.

A single device 500 could sense two or more analytes as well even usingone electrode. If square wave voltammetry is utilized to interrogate theaptamers it is well known that there is an optimum square wave scanningfrequency (typically between 10 and 1000 Hz) for each aptamer, and oftena square wave scanning frequency that results in zero signal gain asanalytes bind with aptamers. This same frequency dependence could beused, for example, to have a first set of aptamers 570, 572, or 574 in adevice 500 (FIGS. 5A-5E) that respond to phenylalanine with a strongestresponse frequency at 30 Hz, and second set of aptamers 570, 572, or 574in a device 500 that respond to cortisol with a strongest responsefrequency at 300 Hz, and the electrode 550 simply modulated in frequencyto measure phenylalanine or cortisol. As a result, a single device canhave two or more aptamer types that enable it to sense two or moreanalytes. Similarly, subsets of aptamers can have different redox tagswith different redox peak potentials, to use applied potential todiscriminate between two or more analytes. Other ways to discriminateaptamer types include electron transfer rates (time), and othervariables in aptamer design that effect their signal gains.

Further, although not described in detail herein, the embodiments of thepresent invention may also be applied to a device that has a pluralityof electrodes used at different times in order to prolong devicelongevity. As described above, electrodes can be passivated withpassivating materials that are exogenous or endogenous, and exogenouspassivation such as polyethyleglycol-terminated passivation can have avery long-lasting resistance to fouling. However, during operation ofthe electrode 250, 450 the passivation can degrade or the electrodeitself could degrade (e.g. become pitted, delaminate, be etched entirelyaway, etc.). Therefore, in embodiments of the present invention aplurality of electrodes can be used at different times inorder toprolong device longevity. For example, one electrode could be used for 3days until it degrades, then a second electrode for 3 days, then a 3rdelectrode for 3 days, for a total device longevity of >1 week.

Although not described in detail herein, other steps which are readilyinterpreted from or incorporated along with the disclosed embodimentsshall be included as part of the invention. The embodiments that havebeen described herein provide specific examples to portray inventiveelements, but will not necessarily cover all possible embodimentscommonly known to those skilled in the art.

EXAMPLES Example 1

With reference to FIGS. 10A and 10B a cortisol binding aptamer wasutilized in a manner similar to that taught in FIG. 6C, where thesignaling aptamer 672 was tagged with methylene blue as a redox tag 640with an aptamer sequence of GTCGTCCCGAGAG [SEQ ID NO.1] and where theanchor aptamer 674 with a sequence ofctctcgggacgacGCCCGCATGTTCCATGGATAGTCTTGACTAgtcgtccc [SEQ ID NO. 2].Electrodes 650, 652 were gold interdigitated electrodes with a 5 μmspacing in between them. The gold electrodes were passivated with anexogenous molecule of mercaptohexanol. No hydrogel 638 was utilized inthis experiment. The sensor solution was buffer solution with 5 μM ofthe aptamers 650, 652 in solution, and a reference electrode of platinumwas used. The device 600 was measured amperometrically vs. a titrationcurve of cortisol as the analyte 19. The results are shown in FIG. 10Aand FIG. 10B (open circles, open diamonds, and solid diamonds), and acontrol experiment with titration of simply adding more cortisol butwithout aptamer in solution is also shown in FIG. 10B (solid circles).The signal gain in Example 1 is as much as 70%, and if the anchoraptamer was made even larger or smaller the signal gain could be tunedto be as much as 200% or more as little as 5% based on the change indiffusion rate of the signaling aptamer to the electrode compared to thesignaling aptamer when it is bound to the anchor aptamer. Signal gain isalso measured above a baseline signal, and changing signaling aptamerconcentration can therefore be used to tune the signal gain.

Example 2

The experiment of Example 1 was repeated but instead of usingmercaptohexanol passivation of the gold electrodes 650, 652, endogenoussmall molecule solutes found in blood or interstitial fluid were allowedto passivate the gold electrode 650, 652. It was found that withoutpassivation background current was very high, but that bothmercaptohexanol and endogeneous solutes were able to adequately reducebackground current and enable sensor operation.

Example 3

Sensors were tested with square wave voltammetry, and redox peaks viavoltammetry were observable with 100 nM of aptamer. Higher aptamerconcentrations only increase the amount of signal and 1 μM, and 10 μMand 100 μM of aptamer were tested as well. Generally, lower aptamerconcentrations were preferred as they reduce device lag times as theyrequire less concentration of analyte to create a change in sensorsignal.

Example 4

Assume a signaling aptamer 672 of 5 kDa and an anchor aptamer 674 of 100kDa, and assume the signaling aptamer when freed from the anchor aptamerhas a diffusion coefficient increase of 4×. Assume the signaling aptameris 98% of the concentration of the anchor aptamer. Assume a simple goldrod electrode 650 that does not necessarily rely on redox recycling asillustrated in FIG. 6A.

According to the Cotrell equation chronoamperometric current isproportional to (D/t){circumflex over ( )}½. Further assume ameasurement of the slope or change in slope of a chronamperometricresponse at 50 ms or greater, such as between 50 ms and 100 ms, which isafter charging current durations and well past the electron transfertime scales of any aptamers that are stuck on or near the electrode 650.Said in another way, chronoamperometric measurement is performed and hasa data measurement window within the chronoamperometric measurementcurve that is after charging currents have dissipated to less than 10%of the current value provided by electron transfer from the aptamers.This particular embodiment brings several potential advantages. First,it produces a 2× (100% signal gain) with binding/unbinding of theanalyte. Secondly, this approach is calibration free. The signal gain(or decrease) due to analyte binding is proportional to the percentageof signaling aptamer that is free from the anchor aptamer. The signalgain can even be invariant to changes in passivation layer 16 thicknessby simply using a small over voltage (for example −0.5V instead of −0.3Vif the redox tag 640 had a peak at −0.3V). Consider an intereferent suchas NADH/NAD+ (663 Da) and which is −20 nM each in serum and therefore 50nM total concentration. The 5 kDa signaling aptamer would have D that is3× greater than NADH, and with the Cotrell equation the currentdifference between NADH and the signaling aptamer would be 3{circumflexover ( )}(½) or 1.73×. So, to have 10× more current from signalingaptamer than NADH/NAD+ it would require 50 nM*10*1.73=865 nM or >100 nMof aptamer, and preferably >1 μM of aptamer. Embodiments can be enabledto be user calibrated, factory calibrated, or calibration free. Usercalibration could for example require a pin-prick blood draw and runningof a conventional assay to measure analyte concentration, and thatconcentration data entered into the software that runs the sensingdevice. Factory calibrated implies that the device requires calibration,but that the calibration is shelf-stable and stable for at least aportion of the use period of the device. Embodiments such as FIG. 6could benefit from factory calibration if calibration free operation isnot possible, and benefit if the passivation layer 648 thickness is keptfairly constant (e.g., using a mercaptohexanol passivation layer 648 orpolyethylene glycol terminated passivation layer 648 that is resistantto fouling).

Although not described in detail herein, other steps which are readilyinterpreted from or incorporated along with the disclosed embodimentsshall be included as part of the invention. The embodiments that havebeen described herein provide specific examples to portray inventiveelements, but will not necessarily cover all possible embodimentscommonly known to those skilled in the art.

What is claimed is:
 1. A device for detecting the presence of, ormeasuring the concentration or amount of, at least one analyte in asample fluid, the device comprising: at least one electrode; a sensorfluid; a plurality of aptamers freely diffusing in the sensor fluid; anda plurality of redox tags associated with at least a subset of aptamersof the plurality of aptamers; wherein the sensor fluid is capable offluidic connection with a sample fluid introduced thereinto; and whereinthe detection or measurement of any analyte may occur through a changein electron transfer from at least one redox tag of the plurality ofredox tags.
 2. The device of claim 1, wherein the device is a continuoussensing device.
 3. The device of claim 1, wherein the device is asingle-use device.
 4. The device of claim 1, further comprising apassivating layer on the at least one electrode.
 5. The device of claim4, wherein the passivating layer includes exogenous molecules.
 6. Thedevice of claim 4, wherein the passivating layer includes molecules thatare endogenous to the sample fluid.
 7. The device of claim 6, wherein asource of the endogenous molecules is the sample fluid.
 8. The device ofclaim 6, wherein a source of the endogenous molecules is the sensorfluid.
 9. The device of claim 1, further comprising at least onemembrane, wherein the membrane separates the sample fluid and the sensorfluid and the membrane retains at least a portion of the aptamers in thesensor fluid.
 10. The device of claim 9, wherein the at least onemembrane has a molecular weight cutoff that is chosen from less than 300Da, less than 1000 Da, less than 3 kDa, less than 10 kDa, less than 30kDa, less than 100 kDa, and less than 300 kDa.
 11. The device of claim9, wherein the change in electron transfer is chosen from greater than5%, greater than 10%, greater than 20%, greater than 50%, greater than100%, and greater than 200%.
 12. The device of claim 1, wherein themeasurement of change in electron transfer is performed usingvoltammetry.
 13. The device of claim 1, wherein the plurality ofaptamers comprise a plurality of signaling aptamers and a plurality ofanchor aptamers.
 14. The device of claim 13, wherein the plurality ofredox tags are bound to the signaling aptamers, but are not bound to theanchor aptamers.
 15. The device of claim 14, wherein the plurality ofanchor aptamers are adapted to bind the analyte, and wherein eachsignaling aptamer of a majority of the plurality of signaling aptamersis bound to a respective anchor aptamer when a majority of anchoraptamers are not bound to any analyte.
 16. The device of claim 14,wherein the plurality of anchor aptamers are adapted to bind theanalyte, and wherein a subset of signaling aptamers dissociates from theanchor aptamers when at least a subset of anchor aptamers bind to anyanalyte.
 17. The device of claim 13, wherein the concentration ofsignaling aptamer is less than the concentration of anchor aptamer. 18.The device of claim 13, wherein the plurality of anchor aptamers areimmobilized to a first material.
 19. The device of claim 18, wherein thefirst material is not freely diffusing in fluid.
 20. The device of claim1, wherein the plurality of redox tags comprises two or more redox tagsper each aptamer of the plurality of aptamers, wherein the distancebetween the two or more redox tags alters depending on the presence ofanalyte.
 21. The device of claim 20, wherein the two or more redox tagsare comprised of identical molecules.
 22. The device of claim 20,wherein the two or more redox tags are comprised of different molecules.23. The device of claim 1, wherein the electrode is coated with at leastone membrane.
 24. The device of claim 1, wherein the concentration ofthe plurality of aptamers is chosen from less than 500 nM, less than 5μM, and less than 50 μM.
 25. The device of claim 1, where each aptamerof the plurality of aptamers has a molecular weight chosen from at least1 kDa, at least 10 kDa, and at least 100 kDa.
 26. The device of claim 1,wherein the change in electron transfer is associated with a conditionchosen from a change in folding pattern of the aptamer, a changing inbinding between two or more aptamers, a change in distance between theredox tag and the electrode, a change in rate of diffusion for the redoxtag to the electrode, a change in electrochemical behavior of the redoxtag, a change in hydrodynamic radius of the aptamer, a change indiffusion coefficient of the aptamer, a change in redox potential of theredox tag, a change in redox current magnitude of the redox tag, and achange in electron transfer rate.
 27. The device of claim 1, wherein thedevice is factory-calibrated.
 28. The device of claim 1, wherein thedevice is calibration-free.
 29. The device of claim 1, wherein thedevice contains two or more types of aptamers for measurement of two ormore analytes.
 30. The device of claim 29, wherein distinguishingbetween measurement of two or more analytes is accomplished via use ofone or more of frequency, potential, and time.
 31. The device of claim1, wherein the at least one electrode further comprises a plurality ofelectrodes, wherein each electrode of the plurality of electrodes is areused at a different time than each of the other electrodes of theplurality of electrodes.
 32. The device of claim 1, wherein themeasurement of change in electron transfer is amperometry.
 33. Thedevice of claim 1, wherein the in electron transfer is due to a changein diffusion coefficient for the aptamers.
 34. The device of claim 1,wherein the at least one electrode is paired with at least a secondelectrode and the distance between the electrodes is chosen from lessthan 50 μm, less than 10 μm, less than 2 μm, and less than 0.4 μm. 35.The device of claim 1, wherein the measurement of change in electrontransfer is chronoamperometry or chronocoulometry.
 36. The device ofclaim 35, wherein the chronoamperometric or chronocoulometricmeasurement is performed and has a data measurement window within themeasurement curve that is after charging currents have dissipated toless than 10% of the current value provided by electron transfer fromthe aptamers.
 37. The device of claim 36, wherein the chronoamperometricor chronocoulometric measurement has a slope and a change in slope andthe slope or change in slope is the measurement of analyte through achange in electron transfer.
 38. A method for detecting the presence of,or measuring the concentration or amount of, at least one analyte in asample fluid, the method comprising: bringing a sample fluid intocontact with at least one aptamer of a plurality of aptamers that arefreely diffusing in a sensor fluid, wherein a plurality of redox tagsare associated with at least a subset of aptamers of the plurality ofaptamers; and detecting or measuring a change in electron transfer fromat least one redox tag of the plurality of redox tags.
 39. The method ofclaim 38, wherein bringing the sample fluid into contact with at leastone aptamer further comprises introducing solutes in the sample fluidinto the sensor fluid.
 40. The method of claim 38, wherein the change inelectron transfer results from a condition chosen from a change infolding pattern of the aptamer, a changing in binding between two ormore aptamers, a change in distance between the redox tag and theelectrode, a change in rate of diffusion for the redox tag to theelectrode, a change in electrochemical behavior of the redox tag, achange in hydrodynamic radius of the aptamer, a change in diffusioncoefficient of the aptamer, a change in redox potential of the redoxtag, a change in redox current magnitude of the redox tag, and a changein electron transfer rate.
 41. The method of claim 38, wherein detectingor measuring a change in electron transfer from at least one redox tagcomprises the use of an electrical measurement technique.
 42. The methodof claim 41, wherein the electrical measurement technique is chosen fromvoltammetry, square wave voltammetry, amperometry, chronoamperometry,coulometry, and chronocoulometry.
 43. The method of claim 42, whereinthe electrical measurement technique is square wave voltammetry.
 44. Themethod of claim 38, wherein detecting or measuring a change in electrontransfer further comprises taking only one measurement.
 45. The methodof claim 38, wherein detecting or measuring a change in electrontransfer further comprises taking multiple separate measurements over adefined time period.